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2021 Т. 51 № 2 / Техника и технология пищевых производств / Food Processing: Techniques and Technology
ISSN 2074-9414 (Print) ISSN 2313-1748 (Online)
https://doi.org/10.21603/2074-9414-2021-2-423-432 Original article
Available online at http://fptt.ru/eng
Callus cultures of Thymus vulgaris and Trifolium pratense as a source of geroprotectors
Lyubov S. Dyshlyuk , Anastasiya M. Fedorova* , Anna I. Loseva , Natalya I. Eremeeva
Kemerovo State University™", Kemerovo, Russia
Received: March 15, 2021
Accepted: May 15, 2021
*e-mail: [email protected] © L.S. Dyshlyuk, A.M. Fedorova, A.I. Loseva, N.I. Eremeeva, 2021
Abstract.
Introduction. Geroprotectors are biologically active substances that inhibit the aging process. Many plant species are natural geroprotectors. For instance, Thymus vulgaris and Trifolium pratense are callus cultures with strong geroprotective properties.
Study objects and methods. The present research featured T. vulgaris and T. pratense grown in vitro on various nutrient media. Their extracts were obtained by aqueous-alcoholic extraction using the following parameters: water-ethanol solvent Se = 30, 50, and 70 %; temperature Te = 30, 50, and 70°C; time Te = 2, 4, and 6 h. The quantitative and qualitative analysis was based on high-performance liquid mass spectrometry, gas mass spectrometry, and thin-layer chromatography.
Results and discussion. The optimal extraction parameters for T. vulgaris were Te = 4 h, Te = 50°C, Se = 70 %, for T. pratense -Te = 6 h, Te = 70°C, Se = 70 %. The chromatography detected flavonoids, phenylpropanoids, simple phenols, higher fatty acids, mono- and sesquiterpenes, and aliphatic hydrocarbons. T. vulgaris appeared to have the highest content of thymol (23.580 ± 1.170 mg/mL); its thymol, apigenin, gallic, chlorogenic, and caffeic components demonstrated geroprotective properties. The extract of T. pratense possessed the highest rutin content (10.05 ± 0.35 mg/mL), and it owed its geroprotective characteristics to rutin, chlorogenic and />-coumaric acids.
Conclusion. The callus cultures of T. vulgaris and T. pratense proved to be promising sources of geroprotective biologically active substances.
Keywords. Extraction, geroprotectors, Thymus vulgaris, Trifolium pratense, biologically active substances
Funding. The research was supported by the Ministry of Science and Higher Education of the Russian Federation (Minobmauka) R0R, project No. FZSR-2020-0006, "Screening of biologically active substances of plant origin with geroprotective properties and development of technology for producing nutraceuticals that inhibit aging".
For citation: Dyshlyuk LS, Fedorova AM, Loseva AI, Eremeeva NI. Callus cultures of Thymus vulgaris and Trifolium pratense as a source of geroprotectors. Food Processing: Techniques and Technology. 2021;51(2):423-432. https://doi.org/10.21603/2074-9414-2021-2-423-432.
https://doi.org/10.21603/2074-9414-2021-2-423-432 Оригинальная статья
УДК 615.322(616-003.725+615.281.9)
http://fptt.ru
Каллусные культуры Thymus vulgaris и Trifolium pratense - перспективные источники активных веществ геропротекторного действия
Л. С. Дышлюк , А. М. Федорова* , А. И. Лосева , Н. И. Еремеева
Кемеровский государственный университет'^, Кемерово Россия
Дата поступления в редакцию: 15.03.2021
Дата принятия в печать: 15.05.2021
*e-mail: [email protected] © Л. С. Дышлюк, А. М. Федорова, А. И. Лосева, Н. И. Еремеева, 2021
Аннотация.
Введение. Естественными источниками биологических активных веществ (БАВ) являются различные виды растений, обладающие геропротекторными свойствами, замедляющие процессы старения. Thymus vulgaris и Trifolium pratense обладают высоким содержанием БАВ. Данная работа посвящена исследованию экстрактов каллусных культур T. vulgaris и T. pratense на наличие веществ геропротекторной направленности.
Объекты и методы исследования. Для получения экстрактов использовались каллусные культуры T. vulgaris и T. pratense, выращенные в условиях in vitro на питательных средах различного состава. Для извлечения веществ выбран метод жидкостной водно-спиртовой экстракции. В качестве растворителя использовалась водно-этанольная смесь (Сэ = 30, 50, 70 %). Также варьировали следующие параметры экстракции: температуру Тэ = 30, 50, 70 °C, время тэ = 2, 4, 6 ч. Количественный и качественный анализ экстрактов каллусных культур T. vulgaris и T. pratense исследовали с применением высокоэффективной жидкостной, газовой с масс-спектрометрией и тонкослойной хроматографии. Результаты и их обсуждение. Для получения экстрактов каллусных культур рекомендованы следующие рабочие параметры: для T. vulgaris - тэ = 4 ч, Тэ = 50 °C, Сэ = 70 %, для T. pratense - тэ = 6 ч, Тэ = 70 °C, Сэ = 70 %. По результатам хроматографического исследования экстрактов установлено наличие флавоноидов, фенилпропаноидов, простых фенолов, высших жирных кислот, моно- и сескитерпенов и алифатических углеводородов. В T. vulgaris наибольшее содержание тимола (23,580 ± 1,170 мг/мл), а в T. pratense наибольшее содержание рутина (10,05 ± 0,35 мг/мл). Из найденных веществ в экстракте T. vulgaris геропротекторной активностью обладают тимол, апигенин, галловая, хлорогеновая и кофейная кислоты; а в T. pratens рутин, хлорогеновая и и-кумаровая кислоты.
Выводы. Каллусные культуры T. vulgaris и T. pratense являются источниками БАВ геропротекторной направленности.
Ключевые слова. Экстракция, геропротекторы, Thymus vulgari, Trifolium pratense, биологические активные вещества
Финансирование. Работа выполнена при финансовой поддержке Министерства науки и высшего образования Российской Федерации (Минобрнауки России)R0R (проект FZSR-2020-0006 «Скрининг биологически активных веществ растительного происхождения, обладающих геропротекторными свойствами, и разработка технологии получения нутрицевтиков, замедляющих старение»).
Для цитирования: Каллусные культуры Thymus vulgaris и Trifolium pratense - перспективные источники активных веществ геропротекторного действия / Л. С. Дышлюк [и др.] // Техника и технология пищевых производств. 2021. Т. 51. № 2. С. 423-432. (На англ.). https://doi.org/10.21603/2074-9414-2021-2-423-432.
Introduction
Environment directly affects human health [1]. Urban environment is a reliable source of various noxious factors [2, 3]. One's health status depends on how well the body has adapted to the environment, while one's functional capabilities are based on the physiological profile, age, and character [4]. Any disease comes from this or that violation of adaptive mechanisms, i.e. when the body fails to resist unfavorable environmental conditions, e.g. air pollution, water contamination, vibration, radiation, noise, electromagnetic, etc. [5-7].
Plant materials can resolve various health issues and inhibit early aging [8]. Plant extracts are the sources of beneficial biological active compounds that can prevent aging, increase life expectancy, improve physical, mental, emotional, and social state, etc. [9-14].
Biologically active compounds of plant origin can be divided into several chemical groups. Glycosides include cardiac glycosides, cyanogenic glycosides, glucosinolates, saponins, and anthraquinone glycosides. Phenolic compounds involve phenolic and hydroxycinnamic acids, stilbenes, flavonoids, and anthocyanins. Tannins are divided into condensed tannins, e.g. large polymers of flavonoids, and hydrolysable tannins, which consist of a monosaccharide nucleus with several catechin derivatives attached. The list of biologically active
compounds also includes mono-, di- and sequiterpenoids, phenylpropanoids, lignans, resins, alkaloids, furocoumarins and naphthodianthrons, proteins, and peptides [15, 16].
Secondary plant metabolites are potential geroprotectors. Their list includes flavonoids, polyphenols, glycosides, tannins, and vitamins. Other compounds that are believed to possess geroprotective properties are rutin, ononin, genistein, rapamycin, carvacrol, resveratrol, apigenin, metformin, terpenene, thymol, gallic acid, isoquercetin, oleanolic acid, /-coumaric acid, and various senolytics. Experiments on mice confirmed that these compounds improve cognitive, neuromuscular, metabolic, and immune systems, inhibit cataracts, sarcopenia, osteoarthritis, osteoporosis, atherosclerosis, Alzheimer's disease, and various age-related tissue dysfunctions [17].
Extraction is the main technological process that produces biologically active compounds from plant raw materials [18]. Extraction process includes three main stages: (1) interaction of plant material with the extractant, (2) destruction of plant cell components, and (3) transfer of solutes to the extractant [19, 20].
Extractants have to be able to penetrate cell walls and selectively dissolve biologically active compounds inside the cell. Therefore, a good extractant has to meet certain requirements, e.g. maximum solubility of active substances; selectivity; high penetrating power;
safety for the human body; volatility, and a low boiling point [21].
All extraction solvents are divided according to polarity. Polar extractants include water, glycerin, etc. They dissolve salts, alkaloids, glycosides, saponins, traglycosides, furocoumarins, organic acids, etc. Aqueous-alcoholic solutions have similar properties. Low-polarity extractants include ethanol, isopropyl, and butyl. They extract salts, alkaloids, flavones, carotenoids, essential oils, pigments, chlorophyll, glycosides, etc. Non-polar extractants include chloroform, hexane, benzene, etc. They extract flavones, essential oils, alkaloids, sapogenins, waxes, fats, etc. Ethanol and water are the most common solvents [22, 23]. As a rule, extraction and isolation of biologically active substances from natural sources follow a well-established procedure: exhaustive extraction (maceration, steam or hydrodistillation, pressing, decoction, infusion, percolation, and Soxhlet extraction); additional chemical processing to isolate the target compounds [13].
Plants are known to synthesize and accumulate secondary metabolites of various phytochemical groups [24, 25]. Callus, suspension, and root cultures are induced for analytical and quantitative comparative analyses of the secondary synthesis of metabolites between plant material and callus, suspension, and root extracts [22, 23].
Among all the medicinal wild plants of the Siberian Federal District, Thymus vulgaris and Trifolium pratense contain the most impressive amount of geroprotective biologically active substances, including such antioxidants as flavonoids, coumarins, etc. [26].
T. vulgaris has antiseptic, antimicrobial, and antioxidant properties [27]. Its extracts are used to treat dyspepsia and gastrointestinal disorders, cough, whooping cough, bronchitis, laryngitis, and tonsillitis, since it contains benzyl alcohol, rutin, apigenin, thymol, gallic acid, luteolin, etc. [28, 29]. T. vulgaris owes its high antimicrobial and antifungal activity to such phenolic compounds as thymol and carvacrol [26]. The yield of essential oil ranges from 0.3 to 6.3% [30]. The content of thymol in the essential oil can reach 60%, which is significantly higher than the content of carvacrol (up to 6%) [31]. The antiseptic effect of thymol is 30 times higher than that of phenol, while its toxic effect is 4 times lower [32]. Phenolic compounds of T. vulgaris can form oxygen free radicals [33].
T. pratense is used as animal feed. This melliferous plant is very popular in agriculture [34]. As a result, its biologically active substances have become focus of constant scientific attention. T. pratense contains some flavonoids, isoflavonoids, and phenolic compounds, e.g. quercetin, rutin, genistein, formononetin, etc. [32]. This plant is used as an antioxidant, antimicrobial and diuretic medicine, as well as a remedy against coronary and nephric edema [34-36].
The research objective was to perform a quantitative and qualitative analysis of callus extracts of T. vulgaris and T. pratense to evaluate their geroprotective prospects.
Study objects and methods
The research featured callus cultures obtained from seeds of Thymus vulgaris and Trifolium pratense grown in vitro. The seeds were washed in soapy water for 30 min and then washed in bidistilled water three times for 20 min. After that, they were treated with 70% ethanol for 1 min and washed three times in bidistilled water for 20 min. Finally, the seeds were washed with 5% sodium hypochlorite solution for 50 min and washed three times in bidistilled water for 20 min [37]. After sterilization, the seeds were planted on agar media. Table 1 shows the composition of the nutrient media.
The first seedlings of T. pratense appeared on week 1-2, and those of T. vulgaris - on week 4-5. The experiment featured sterile seedlings that were 2-5 weeks old. The explants were cut into pieces and planted in agar media. The first calli appeared during 14 days of cultivation. Further callus formation involved Murashige-Skoog (MS), Gamborg (B5), and Schenck Hildebrandt (SH) mineral bases with casein hydrolyzate (0.5 g/L), inositol (0.1 g/L), 3% sucrose or glucose, and 2% agar. The media varied in the composition of growth regulators, which included indoleacetic acid, 2,4-dichlorophenoxyacetic acid, kinetin, and 6-benzylaminopurine (Table 2). The explants were incubated for 25 days.
The primary callus was separated from the remains of the explants and transferred to a fresh nutrient medium to grow for 4-5 weeks.
The callus cultures were extracted by the standard method of liquid aqueous-alcoholic extraction using ethanol (State Standard 5962-2013. Rectified ethyl
Table 1. Nutrient media for seedlings of Thymus vulgaris and Trifolium pratense
Components Per 1 l of distilled water
Thymus vulgaris Trifolium pratense
B5 macrosalts (20*)*, mL 50.00 50.00
B5 microsalts (20*)*, mL 10.00 10.00
Fe-EDTA, mL 5.00 5.00
Sucrose, g 30.00 30.00
Nicotinic acid, mg 1.00 0.50
Pyridoxine, mg 1.00 0.10
Thiamine, mg 10.00 0.10
Inositol, mg 100.00 100.00
6-benzylaminopurine, mg - 1.00
Activated carbon, g - 20.00
Agar, g 20.00 20.00
pH 5.4-6.0 5.4-6.0
* see T.A. Murashige [38].
Table 2. Mineral and hormone composition of the culture medium for the cultivation of callus cultures of Thymus vulgaris and Trifolium pratense
Components Per 1 mL of distilled water
Thymus vulgaris Trifolium pratense
Medium 1 Medium 2 Medium 3 Medium 1 Medium 2 Medium 3
MS macrosalt (20x)**, mL 50.00 50.00 50.00 50.0 50.00 50.00
MS microsalt (20x)**, mL 1.00 1.00 1.00 10.0 10.00 10.00
Fe-EDTA, mL 5.00 5.00 5.00 5.00 5.00 5.00
Thiamine, mg 0.10 0.10 0.10 10.00 10.00 10.00
Pyridoxine, mg 0.50 0.50 0.50 1.00 1.00 1.00
Nicotinic acid, mg 0.50 0.50 0.50 1.00 1.00 1.00
Sucrose, g 30.00 30.00 30.00 30.00 30.00 30.00
Casein hydrolyzate, mg 500.00 500.00 500.00 - - -
Inositol, mg 100.00 100.00 100.00 100.00 100.00 100.00
Kinetin, mg - 2.00 1.00 - 1.00 2.00
6-benzylaminopurine, mg - 0.50 - 0.05 - 0.10
Indoleacetic acid, mg - - 2.00 1.00 - 2.00
Peoxyethanolic acid, mg - 3.00 - - - -
2,4-dichlorophenoxyacetic acid, mg - - - - 1.00 2.00
Agar, g 20.00 20.00 20.00 20.00 20.00 20.00
pH 5.4-6.0 5.4-6.0 5.4-6.0 5.4-6.0 5.4-6.0 5.4-6.0
** see O.L. Gamborg [39].
alcohol from edible raw material. Specifications) and distilled water (State Standard 6709-72. Distilled water. Specifications) as solvent [41]. The water was purified in a BS brand bidistiller (Labinvest, Russia).
The extraction parameters were as follows: extraction temperature - 30, 50, and 70°C; time - 2, 4, and 6 h, ethanol concentration - 30, 50, and 70%.
The callus cultures of T. vulgaris and T. pratense were dried and ground in an LZM-1M rotary mill (Olis, Russia). The extraction samples weighed 3.000 ± 0.001 g.
The volume of ethanol was 260 mL. The extraction was carried out in a water bath (Elmasonic S60H, Germany) with an ascending refrigerator at a given temperature, time, and ethanol concentration. The obtained extracts T. vulgaris and T. pratense were stored at room temperature in the dark.
The antioxidant activity of the extracts was determined to define the total biologically active substances. It was expressed as the content of the sum of biologically active substances of a reducing nature in terms of quercetin
Table 3. Antioxidant activity of Thymus vulgaris callus extracts
Tem- Volume fraction of ethanol, %
pera- 30 50 70
ture, °C Extraction time, 2 h
30 0.2080 ± 0.0020 0.1250 ± 0.0012 0.1640 ± 0.0029
50 0.2250 ± 0.0023 0.1920 ± 0.0017 0.2240 ± 0.0030
70 0.1980 ± 0.0011 0.2180 ± 0.0030 0.2270 ± 0.0033
Extraction time, 4 h
30 0.1576 ± 0.0016 0.2182 ± 0.0022 0.2048 ± 0.0036
50 0.1940 ± 0.0026 0.2134 ± 0.0030 0.2956 ± 0.0047
70 0.2687 ± 0.0031 0.2430 ± 0.0050 0.2873 ± 0.0050
Extraction time, 6 h
30 0.2253 ± 0.0055 0.1939 ± 0.0030 0.2831 ± 0.0060
50 0.1811 ± 0.0046 0.2584 ± 0.0036 0.2752 ± 0.0049
70 0.1711 ± 0.0013 0.2388 ± 0.0050 0.2643 ± 0.0030
Table 4. Antioxidant activity of Trifolium pratense callus extracts
Tem- Volume fraction of ethanol, %
pera- 30 50 70
ture, °C Extraction time, 2 h
30 0.1906 ± 0.0014 0.1464 ± 0.0010 0.1630 ± 0.0018
50 0.2122 ± 0.0030 0.2143 ± 0.0045 0.1703 ± 0.0040
70 0.1475 ± 0.0010 0.1847 ± 0.0032 0.1942 ± 0.0021
Extraction time, 4 h
30 0.1741 ± 0.0030 0.1743 ± 0.0027 0.1460 ± 0.0040
50 0.1494 ± 0.0022 0.1852 ± 0.0016 0.1737 ± 0.0045
70 0.1046 ± 0.0010 0.1057 ± 0.0011 0.2392 ± 0.0056
Extraction time, 6 h
30 0.1621 ± 0.0032 0.1329 ± 0.0010 0.1636 ± 0.0010
50 0.1419 ± 0.0018 0.1637 ± 0.0034 0.1813 ± 0.0021
70 0.1547 ± 0.0010 0.1386 ± 0.0026 0.1918 ± 0.0030
50-
40-
20-
10-
г r
<*
J^J
JLvJ / J....L
D.0
2.5 5.0 7.5 id
.0 12.5 15.0
17.5 2d.0 22.5 25.0
27.5
3d.0
32.5
Figure 1. Chromatogram of the aqueous-alcoholic composition of Thymus vulgaris callus cultures: 1 - gallic acid; 2 -oleanolic acid; 3 - chloric acid; 4 - ursolic acid; 5 - apigenin-7-glucoside; 6 - caffeic acid;
7 - apigenin; 8 - carvacrol; 9 - thymolic acid
in 1 mL of the extract by the method developed by T.V. Maksimova [43].
The experiment involved a liquid chromatograph (Shimadzu LC-20 Prominence, Japan) with a Shimadzu SPD-20-MA diode array detector and a RID-1 OA refractometric detector, a chromatographic column Kromasil 5 (im C18, 250x4.6 mm, a Guard Column Security Guard Gartridge (CI8) Phenomenex (USA) with injection volume 20 (j,L. The column temperature was 30°C; the elution mode was isocratic; the mobile phase consisted of AcCN:isopropyl alcohol:H90-H3P04 (20:5:75, pH 3.5).
Gas chromatography with mass spectrometry (GC-MS) and thin layer chromatography (TLC) were carried out at the same time as HPLC [44].
The analysis of biologically active substances involved Sorbfil PTS-AF-A TLC plates. The obtained extract was applied to the start line, dried, and placed in a chromatographic chamber filled with a mix of w-butanol, acetic acid, and water at a ratio of 60:15:25. After lOmin, a 25% solution of phosphoric-tungstic acid was added at 95°C. The densitometric analysis of the plate was performed using a Handycam HDR-CX405 densitometer with a Sony photofixation system (OOO IMID, Russia).
The T. vulgaris callus extracts underwent a GC-MS using a 30 m column with an inner diameter of 0.25 mm and helium as a carrier gas. The main parameters for GC-MS were as follows: carrier gas flow rate -1.4 mL/min; interface temperature - 280°C; injector temperature - 240°C; column temperature - 100-270°C; volume of the injected sample - 3 ^L. The sample was introduced without dividing the carrier gas flow.
Results and discussion
Nutrient medium 2 proved optimal for the callusogenesis of Thymus vulgaris, which included
the following growth hormones: kinetin - 2 mg, 6-benzylaminopurine - 0.5 mg, peoxyethanolic acid -
3 mg. When the callus culture of Trifolium pratense was cultivated on nutrient medium 2, which contained 1 mg of kinetin and 2,4-dichlorophenoxyacetic acid, the callus growth was slow. Nutrient medium 3 proved optimal for T. pratense callus culture: it contained the following growth hormones: kinetin - 2 mg, 6-benzylaminopurine - 0.1 mg, indoleacetic acid - 2 mg, and 2.4- dichlorophenoxyacetic acid - 2 mg.
Tables 3 and 4 show the total content of biologically active substances in terms of quercetin in 1 mL of the extract under different extraction conditions.
The obtained data demonstrated in Tables 3 and
4 made it possible to recommend the following optimal extraction parameters for T. vulgaris: xe - 4 h, Te - 50°C,
5 - 70%; for T. pratense: x - 6 h, T - 70°C, - 70%.
e 7 r e'e 7 e
After establishing the optimal extraction parameters, the next step was to analyze the qualitative and quantitative composition of biologically active substances in aqueous-alcoholic extracts.
Table 5. Component composition of Thymus vulgaris callus extract
Peak Retention time, min Component Quantitative content, mg/mL
1 5.162 gallic acid 5.720 ± 0.320
2 6.610 oleanolic acid 5.290 ± 0.400
3 6.910 chloric acid 1.920 ± 0.300
4 7.240 ursolic acid 2.200 ± 0.200
5 8.860 apigenin-7-glucoside 11.460 ±0.760
6 12.240 caffeic acid 18.930 ± 0.880
7 19.980 apigenin 3.180 ± 0.320
8 22.045 carvacrol 8.170 ± 0.490
9 23.930 thymolic acid 23.580 ± 1.170
(xl ,000,000)
TIC .4: N N
*
3 OD 30 KP 0) N <3 CÖ *
\ .2- V. N 1®- N 00 fro sr 1 pjol T 10 si J mi 4j ¿Ü ra 0
5.0 7.5 10,0 12,5 15,0 17,5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 425 45.0 475 50.0 525 55.0 57.5 60.0 62.5
Figure 2. GC-MS chromatogram of Thymus vulgaris callus culture fraction
Figure 1 shows the results of HPLC analysis for T. vulgaris callus extract, while Table 5 demonstrates the results of the qualitative and quantitative analysis of biologically active substances.
The HPLC analysis (Fig. 1, Table 5) of T. vulgaris callus extracts revealed that the samples contained flavonoids, phenylpropanoids, and simple phenols.
Figure 2 illustrates the GC-MS analysis of T. vulgaris callus extract and displays other individual biologically active substances.
The GC-MS analysis of the composition of biologically active substances in the aqueous-alcoholic callus extracts of T. vulgaris revealed the following composition: mono-and seskyterpenes - tricyclene, thuyenne, terpene, cubeben, verbenone, verbenol, eucalyptol, linalool, bourbonene, borneol, isoborneol, caryophyllene, kadinen, farnesene, cadinol; higher fatty acids - 3-ketopentatriacontanic H-heneicosan; aliphatic hydrocarbons - heptadecane,
nonadecan, heneicosan, heptenol, octanol, 1-dodecanol, 3-octanone (Fig. 2).
The extracts of T. pratense callus culture underwent HPLC and TLC chromatography. Figure 3 demonstrates the HPLC chromatogram.
The HPLC analysis revealed biologically active substances that belong to phenolcarboxylic acids: ^-coumaric acid, />-coumaroyl-3-quinic acid, and chlorogenic acid; flavones: rutin, isoquercitrin; isoflavones: daidzein, genistein. Table 6 demonstrates the qualitative and quantitative HPLC analysis of the sample.
The content of biologically active substances in T. pratense samples changed depending on the extraction method, which was the main peculiarity of this extract. After ultrasonic extraction, the amount of flavanoids was 2.13%, isoflavonoids - 4.42%; after heat maceration, the yield of flavonoids was 1.64%, isoflavonoids - 3.24% [40, 42]. Figure 4 shows the densitogram of the TLC analysis of the Trifolium pratense callus extract.
Table 6. Components of Trifolium pratense callus extract
Peak Retention time, min Component Quantitative content, mg/mL
1 4.49 rutin 10.05 ± 0.35
2 6.15 chlorogenic acid 7.29 ± 0.42
3 7.25 />-coumaroylquinic acid 23.10 ± 0.74
4 9.03 />-coumaric acid 1.26 ± 0.10
5 9.70 isoquercetrin 1.55 ± 0.08
6 10.30 biochanin A 8.89 ± 0.35
7 13.50 ononin 2.51 ± 0.20
8 13.97 daidzein 1.36 ± 0.07
9 17.20 genistein 9.05 ± 0.13
10 47.01 melilotic acid 3.61 ± 0.19
MAU
(
J DU о Л
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 «D.0 45.0 50.0 55.0 min
Figure 3. HPLC chromatogram of Trifolium pratense callus extract: 1 - rutin; 2 - chlorogenic acid; 3 - p-coumaroylquinic acid; 4 - p-coumaric acid; 5 - isoquercetrin; 6 - biochanin A; 7 - ononin; 8 - daidzein; 9 - genistein; 10 - melilotic acid
Figure 4. Densitogram of Trifolium pratense callus extract under TLC separation on a Sorbfil plate: R/ 0.1 - quercetin-3-O-rutinoside (rutin); R/ 0.53 - chlorogenic acid; R/ 0.95 - isoquercitrin
The TLC analysis showed that the T. pratense callus extracts contained such biologically active substances as quercetin-3-O-rutinoside (rutin), chlorogenic acid, and isoquercitrin.
Based on scientific publications and the chromatography performed, the T. vulgaris and T. pratense callus extracts proved to contain secondary metabolites with geroprotective properties. The callus culture of T. vulgaris contained gallic, oleanolic, chlorogenic, and caffeic acids, apigenin, carvacrol, thymol, terpene, verbenone, verbenol, isoborneol, caryophyllene, kadinen, farnesene, and cadinol [45-47]. The callus culture of T. pratense contained such geroprotectors as rutin, chlorogenic, and /-coumaric acid [48-51].
Conclusion
The present research featured the quantitative and qualitative content of biologically active substances in aqueous-alcoholic extracts of callus cultures of Thymus vulgaris and Trifolium pratense. The optimal extraction parameters for T. vulgaris callus culture were as follows: t - 4 h, T - 50°C, S - 70%; for T. pratense: t - 6 h,
e'e 7 e 7 r e
T - 70°C, S - 70%.
ee
The qualitative and quantitative analyses of biologically active substances in the aqueous-alcoholic callus extracts of T. vulgaris and T. pratense were based on chromatography. The HPLC test revealed gallic, oleanolic, chlorogenic, and ursolic acids, apigenin-7-glucoside, caffeic acid, apigenin, carvacrol, and thymol in the T. vulgaris callus
extracts. The selected extraction parameters resulted in a high yield of thymol (23.580 ± 1.170 mg/mL). The GC-MS analysis revealed mono- and sesquiterpene (tricyclene, thujene, terpenene, cubebene, verbenone, verbenol, eucalyptol, linalool, bourbonene, borneol, isoborneol, caryophyllene, kadinene, farnesene, and cadinol), higher fatty 3-ketopentatriacontanic acid, aliphatic hydrocarbons (heptadecane, nonadecane, heneicosan, heptenol, octanol, 1-dodecanol, and 3-octanone).
As for the T. pratense callus extracts, the HPLC analysis revealed rutin, chlorogenic acid, /-coumaroyl-3-quinic acid, /-coumaric acid, isoquercetrin, biochanin A, ononin, daidzein, genistein, and melilotic acid. The selected extraction parameters produced a high yield of rutin (10.05 ± 0.35 mg/mL). According to the TLC chromatography, the T. pratense callus extracts contained rutin, chlorogenic acid, and isoquercitrin.
Therefore, the callus cultures of T. vulgaris and T. pratense proved to be sources of geroprotective biologically active substances.
Contribution
The authors are equally responsible for the information published in this article and any possible cases of plagiarism.
Conflict of interest
The authors declare no conflict of interests regarding the publication of this article.
References
1. Wallace DR, Buha A, Powell JJ, Tsatsakis A. Editorial overview: The environment and man: A Study in mechanistic toxicology. Current Opinion in Toxicology. 2020;19. https://doi.org/10.1016/j.cotox.2020.03.007.
2. Vesnina A, Prosekov A, Kozlova O, Atuchin V. Genes and eating preferences, their roles in personalized nutrition. Genes. 2020;11(4). https://doi.org/10.3390/genes11040357.
3. Dyshlyuk L, Babich O, Prosekov A, Ivanova S, Vasilchenco N, Atuchin V, et al. Antimicrobial potential of ZnO, TiO2 and SiO2 nanoparticles in protecting building materials from biodegradation. International Biodeterioration and Biodegradation. 2020;146. https://doi.org/10.10167j.ibiod.2019.104821.
4. Kumar P, Druckman A, Gallagher J, Gatersleben B, Allison S, Eisenman TS, et al. The nexus between air pollution, green infrastructure and human health. Environment International. 2019;133. https://doi.org/10.1016/j.envint.2019.105181.
5. Jacobson TA, Kler JS, Hernke MT, Braun RK, Meyer KC, Funk WE. Direct human health risks of increased atmospheric carbon dioxide. Nature Sustainability. 2019;2(8):691-701. https://doi.org/10.1038/s41893-019-0323-1.
6. ClaBen T, Bunz M. Contribution of natural spaces to human health and wellbeing. Bundesgesundheitsblatt -Gesundheitsforschung - Gesundheitsschutz. 2018;61(6):720-728. https://doi.org/10.1007/s00103-018-2744-9.
7. Asyakina LK, Fotina NV, Izgarysheva NV, Slavyanskiy AA, Neverova OA. Geroprotective potential of in vitro bioactive compounds isolated from yarrow (Achilleae millefolii L.) cell cultures. Foods and Raw Materials. 2021;9(1):126-134. https:// doi.org/10.21603/2308-4057-2021-1-126-134.
8. Dmitrieva AI, Belashova OV, Milenteva IS, Ivanova SA, Prosekov AYu. Assessment of the content of heavy metals in medicinal plants of genus Trifolium from the growing area on the example of the Siberian Federal District. International Journal of Pharmaceutical Research. 2020;12(3):1880-1893. https://doi.org/10.31838/ijpr/2020.12.03.262.
9. Vardanyan LR, Vardanyan RL, Galstyan AG, Atabekyan LV. Kinetics of the combined anti-oxidant action of the extractions of herb material and their mixtures. Proceedings of Voronezh State University. Series: Chemistry. Biology. Pharmacy. 2019;(4):5-12. (In Russ.).
10. Vesnina AD, Dmitrieva AI, Asyakina LK, Velichkovich NS, Prosekov AYu. Relevance of the use of plant extracts in the creation of functional products that have a geroprotective effect. International Journal of Pharmaceutical Research. 2020;12(3):1865-1879. https://doi.org/10.31838/ijpr/2020.12.03.261.
11. Fotina NV, Dmitrieva AI, Milenteva IS, Prosekov AYu. Optimization of parameters for extracting biologically active substances Medicago sativa L. International Journal of Pharmaceutical Research. 2020;12(3):1857-1864. https://doi. org/10.31838/ijpr/2020.12.03.260.
12. Bubaloa MC, Vidovic S, Redovnikovic IR, Jokicc S. New perspective in extraction of plant biologically active compounds by green solvents. Food and Bioproducts Processing. 2018;109:52-73. https://doi.org/10.1016/j.fbp.2018.03.001.
13. Bilal M, Iqbal HMN. Biologically active macromolecules: Extraction strategies, therapeutic potential and biomedical perspective. International Journal of Biological Macromolecules. 2020;151:1-18. https://doi.org/10.1016/j.ijbiomac.2020.02.037.
14. Chupakhin E, Babich O, Krasavin M, Prosekov A, Asyakina L. Spirocyclic motifs in natural products. Molecules. 2019;24(22). https://doi.org/10.3390/molecules24224165.
15. Kumar M, Dahuja A, Tiwari S, Punia S, Tak Y, Amarowicz R, et al. Recent trends in extraction of plant bioactives using green technologies: A review. Food Chemistry. 2021;353. https://doi.org/10.1016/j.foodchem.2021.129431.
16. Gorguj A, Genjdag E, Yilmaz FM. Bioactive peptides derived from plant origin by-products: Biological activities and techno-functional utilizations in food developments - A review. Food Research International. 202;136. https://doi.org/10.1016/). foodres.2020.109504.
17. Moskalev A, Chernyagina E, Kudryavtseva A, Shaposhnikov M. Geroprotectors: A unified concept and screening approaches. Aging and Disease. 2017;8(3):354-363. https://doi.org/10.14336/AD.2016.1022.
18. Azwanida NN. A review on the extraction methods use in medicinal plants, principle, strength and limitation. Medicinal and Aromatic Plants. 2017;4(3). https:Zdoi.org/10.4172/2167-0412.1000196.
19. Ameer K, Shahbaz HM, Kwon J-H. Green extraction methods for polyphenols from plant matrices and their byproducts: A Review. Comprehensive Reviews in Food Science and Food Safety. 2017;16(2):295-315. https://doi.org/10.1111/1541-4337.12253.
20. Tyskiewicz K, Konkol M, Roj E. The application of supercritical fluid extraction in phenolic compounds isolation from natural plant materials. Molecules. 2018;23(10). https://doi.org/10.3390/molecules23102625.
21. Leonova MV, Klimochkin YuN. Ehkstraktsionnye metody izgotovleniya lekarstvennykh sredstv iz rastitel'nogo syr'ya [Extraction methods in the production of medicinal products from plant raw materials]. Samara: Samara State Technical University; 2012. 118 p. (In Russ.).
22. Lozano-Grande MA, Gorinstein S, Espitia-Rangel E, Davila-Ortiz G, Martinez-Ayala AL. Plant sources, extraction methods, and uses of squalene. International Journal of Agronomy. 2018;2018. https://doi.org/10.1155/2018/1829160.
23. Gonjalves S, Romano A. Green approaches for the extraction of bioactives from natural sources for pharmaceutical applications. In: Inamuddin, Boddula R, Ahamed MI, Asiri A, editors. Green sustainable process for chemical and environmental engineering and science. Solvents for the pharmaceutical industry. Elsevier; 2020. pp. 249-267. https://doi.org/10.1016/B978-0-12-821885-3.00013-X.
24. Jacoby RP, Koprivova A, Kopriva S. Pinpointing secondary metabolites that shape the composition and function of the plant microbiome. Journal of Experimental Botany. 2021;72(1):57-69. https://doi.org/10.1093/jxb/eraa424.
25. Harhaun R, Kunik O, Saribekova D, Lazzara G. Biologically active properties of plant extracts in cosmetic emulsions. Microchemical Journal. 2020;154. https://doi.org/10.10167j.microc.2019.104543.
26. Bhattacharya A. High-temperature stress and metabolism of secondary metabolites in plants. In: Bhattacharya A, editor. Effect of high temperature on crop productivity and metabolism of macro molecules. Academic Press; 2019. pp. 391-484. https://doi.org/10.1016/B978-0-12-817562-0.00005-7.
27. Kalashnikova EA, Zaytseva SM, Doan TT, Kirakosyan RN. Study of biological activity of extracts obtained from microclonal medicinal plants of different taxonomic groups in vitro. Veterinary, Zootechnics and Biotechnology. 2018;(12):50-58. (In Russ.).
28. Leite KCDS, Garcia LF, Lobön GS, Thomaz DV, Moreno EKG, Carvalho MFD, et al. Antioxidant capacity evaluation of dried herbal extracts: an electroanalytical approach. Revista Brasileira de Farmacognosia. 2018;28(3):325-332. https://doi. org/10.1016/j.bjp.2018.04.004.
29. Pavlic B, Sojic B, Teslic N, Putnik P, Kovacevic DB. Extraction of bioactive compounds and essential oils from herbs using green technologies. In: Galanakis CM, editor. Aromatic herbs in food. Bioactive compounds, processing, and applications. Academic Press; 2021. pp. 233-262. https://doi.org/10.1016/B978-0-12-822716-9.00007-X.
30. Manjare SD, Dhingra K. Supercritical fluids in separation and purification: A review. Materials Science for Energy Technologies. 2019;2(3):463-484. https://doi.org/10.1016/j.mset.2019.04.005.
31. Freitas IR, Cattelan MG. Antimicrobial and antioxidant properties of essential oils in food systems - An overview. In: Holban AM, Grumezescu AM, editors. Microbial contamination and food degradation. A volume in handbook of food bioengineering. Academic Press; 2018. pp. 443-470. https://doi.org/10.1016/B978-0-12-811515-2.00015-9.
32. Malik NR, Yadav KC, Verma A. Optimization of process parameters in extraction of thyme oil using response surface methodology (RSM). International Journal of Science, Engineering and Technology. 2016;4(1):248-256.
33. Sojic B, Tomovic V, Kocic-Tanackov S, Kovacevic DB, Putnik P, Mrkonjic Z, et al. Supercritical extracts of wild thyme (Thymus serpyllum L.) by-product as natural antioxidants in ground work parties. LWT. 2020;130. https://doi.org/10.1016/). lwt.2020.109661.
34. Bendif H, Adouni K, Miara MD, Baranauskiene R, Kraujalis P, Venskutonis PR, et al. Essential oils (EOs), pressurized liquid extracts (PLE) and carbon dioxide supercritical fluid extracts (SFE-CO2) from Algerian Thymus munbyanus as valuable sources of antioxidants to be used on an industrial level. Food Chemistry. 2018;260:289-298. https://doi.org/10.1016/]. foodchem.2018.03.108.
35. Mozdzen K, Barabasz-Krasny B, Stachurska-Swakon A, Zandi P, Pula J, Wang YS, et al. Allelopathic interaction between two common meadow plants: Dactylis glomerata L. and Trifolium pratense L. Biologia. 2020;75(5):653-663. https:// doi.org/10.2478/s11756-020-00438-6.
36. Novoselov MYu, Starshinova OA, Drobysheva LV. The possibility of using self-compatible forms of meadow clover (Trifolium pratense L.) in breeding to increase seed productivity. IOP Conference Series: Earth and Environmental Science. 2021;663(1). https://doi.org/10.1088/1755-1315/663/1Z012016.
37. Erkoyuncu MT, Yorgancilar M. Optimization of callus cultures at Echinacea purpurea L. for the amount of caffeic acid derivatives. Electronic Journal of Biotechnology. 2021;51:17-27. https://doi.org/10.1016/j.ejbt.2021.02.003.
38. Murashige T, Scoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiology Plantarum. 1962;15(3):473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x.
39. Gamborg OL, Miller RA, Ojima O. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research. 1968;50(1):151-158. https://doi.org/10.1016/0014-4827(68)90403-5.
40. Andreeva VYu, Kalinkina GI, Poluektova TV, Gulyaeva VA. The comparative study of phenolic compounds in Trifolium L. species in Siberia. Chemistry of Plant Raw Material. 2018;(1):97-104. (In Russ.). https://doi.org/10.14258/jcprm.2018011846.
41. Nguen TSh, Kaukhova IE, Sorokin VV. Vybor metoda ehkstragirovanii flavonoidov iz travy klevera lugovogo [Selecting the method for extracting flavonoids from meadow clover]. Garmonizatsiya podkhodov k farmatsevticheskoy razrabotke: Sbornik tezisov II Mezhdunarodnoy nauchno-prakticheskoy konferentsii [Harmonization of approaches to pharmaceutical development: Proceedings of the II International Scientific and Practical Conference]; 2019; Moscow. Moscow: RUDN University; 2019. p. 204-206. (In Russ.).
42. Popova OS, Skrypnik LN. Comparative characteristic of the efficiency of various extraction methods of polyphenols from plants of the family Lamiaceae. Advances in Current Natural Sciences. 2017;(6):34-38. (In Russ.).
43. Maksimova TV, Nikulina IN, Pakhomov VP, Shkarina EI, Chumakova ZV, Arzamastsev AP. Method for determining antioxidation activity. Patent RU 2170930AC1. 2001.
44. Waksmundzka-Hajnos M, Sherma J, Kowalska T. Thin layer Chromatography in phytochemistry. Boca Raton: CRC Press; 2008. 896 p. https://doi.org/10.1201/9781420046786.
45. Gedikolu A, Sökmen M, Qivit A. Evaluation of Thymus vulgaris and Thymbra spicata essential oils and plant extracts for chemical composition, antioxidant, and antimicrobial properties. Food Science and Nutrition. 2019;7(5):1704-1714. https:// doi.org/10.1002/fsn3.1007.
46. Bazarnova YuG, Ivanchenko OB. Investigation of the composition of biologically active substances in extracts of wild plants. Problems of Nutrition. 2016;85(5):100-107. (In Russ.).
47. Dauqan EMA, Abdullah A. Medicinal and functional values of thyme (Thymus vulgaris L.) herb. Journal of Applied Biology and Biotechnology. 2017;5(2):17-22. https://doi.org/10.7324/JABB.2017.50203.
48. Hanganu D, Benedec D, Vlase L, Olah N, Damian G, Silaghi-Dumitrescu R, et al. Polyphenolic profile and antioxidant and antibacterial activities from two trifolium species. Farmacia. 2017;65(3):449-453.
49. Erta§ A, Boga M, Ha^imi N, Yilmaz MA. Fatty acid and essential oil compositions of Trifolium angustifolium var. angustifolium with antioxidant, anticholinesterase and antimicrobial activities. Iranian Journal of Pharmaceutical Research. 2015;14(1):233-241.
50. Chiriac ER, Chi^escu CL, Borda D, Lupoae M, Gird CE, Geana E-I, et al. Comparison of the polyphenolic profile of Medicago sativa L. and Trifolium pratense L. sprouts in different germination stages using the UHPLC-Q exactive hybrid quadrupole Orbitrap high-resolution mass spectrometry. Molecules. 2020;25(10). https://doi.org/10.3390/Molecules25102321.
51. Zeb A, Hussain A. Chemo-metric analysis of carotenoids, chlorophylls, and antioxidant activity of Trifolium hybridum. Heliyon. 2020;6(1). https://doi.org/10.1016/j.heliyon.2020.e03195.
